Digestibility of nutrients in growing–finishing pigs is affected by Aspergillus niger phytase, phytate and lactic acid levels

Livestock Production Science 58 (1999) 119–127

Digestibility of nutrients in growing–finishing pigs is affected by Aspergillus niger phytase, phytate and lactic acid levels 2. Apparent total tract digestibility of phosphorus, calcium and magnesium and ileal degradation of phytic acid a, a a a b Paul A. Kemme *, Age W. Jongbloed , Zdzisław Mroz , Jan Kogut , Anton C. Beynen b

a Institute for Animal Science and Health ( ID-DLO), P.O. Box 65, 8200 AB Lelystad, The Netherlands Department of Large Animal Medicine and Nutrition, Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.152, 3508 TD Utrecht, The Netherlands

Received 18 March 1998; accepted 3 November 1998

Abstract In growing–finishing pigs, the effects of supplemental microbial phytase, lactic acid and Na phytate in a maize-soybean meal based diet on the apparent total tract digestibility (ATTD) of ash, total P, Ca and Mg and the ileal degradation (AID) of phytic acid were studied. The experimental design was a 2 3 2 3 2 factorial arrangement plus a positive control treatment. Six crossbred castrates of 37 kg initial BW, fitted with steered ileo-caecal valve cannulas were used during six collection periods. The dietary treatments consisted of Aspergillus niger phytase (Natuphos  ; 0 or 900 FTU kg 21 ), sodium phytate (0 or 1.5 g P kg 21 ) or lactic acid (0 or 30 g kg 21 ). The positive control diet was supplemented with 1.0 g P kg 21 from monocalcium phosphate monohydrate (MCP). The feeding level was 2.3 times maintenance requirement for energy (418 kJ MEW 20.75 ). Estimates of AID and ATTD were calculated using Cr 2 O 3 as a marker. The addition of MCP to the diet as the only variable had no effect on the AID of phytic acid and the ATTD of ash, Ca and Mg, but enhanced total P ATTD. Both microbial phytase and lactic acid enhanced the ATTD of ash, Ca and Mg and the AID of phytic acid, but there was no interaction. The ATTD of total P was increased by the combination of microbial phytase and lactic acid to a greater extent than was calculated as the sum of the stimulatory effects of the single additions. It is hypothesized that lactic acid delays gastric emptying, which prolongs the action of phytase in the stomach at its optimum pH. When Na phytate was added to the diets, total P ATTD was enhanced, possibly reflecting efficient phytate hydrolysis by intrinsic phytase.  1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Pigs; Phytase; Acidification; Minerals; Digestibility

1. Introduction Plant ingredients used to formulate diets for pigs contain poorly soluble salts of phytic acid that are *Corresponding author. Tel.: 1 31-320-237-324; fax: 1 31320-237-320. E-mail address: [email protected] (P.A. Kemme)

practically unavailable for the pig (Jongbloed, 1987; Cromwell, 1992). Phytic acid binds approximately two thirds of intrinsic phosphorus in vegetal feedstuffs (Cosgrove, 1980), and it may form insoluble complexes with dietary di- and trivalent cations (Erdman, 1979; Maga, 1982). In consequence, bioavailability of these minerals may be reduced. The addition of phytase to pig diets does not only

0301-6226 / 99 / $ – see front matter  1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S0301-6226( 98 )00202-4

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render phytate P available for absorption ¨ (Dungelhoef and Rodehutscord, 1995; Jongbloed et al., 1996), but also enhances the digestibility of calcium and magnesium (Jongbloed et al., 1996). Dietary acidification may also stimulate the digestion of minerals (Kirchgeßner and Roth, 1980; Ravindran and Kornegay, 1993). A large portion of gastric digesta leaves the pig stomach shortly after feeding and has a pH that is too high for optimal microbial phytase action (Jongbloed et al., 1992). Feed acidification may reduce the rate of gastric emptying (Mayer, 1994), which could favour the action of phytase. It was thus hypothesized that dietary acidification and microbial phytase may have a synergistic effect on mineral digestibility. To test this hypothesis we determined the apparent total tract digestibility (ATTD) of ash, Ca, Mg and P and the gastro-ileal degradation (AID) of phytic acid in growing–finishing pigs which were fed a maize-soybean meal diet with or without added microbial phytase, Na phytate and lactic acid.

2. Material and methods The animals and their housing have been described by Kemme et al. (1999).

2.1. Experimental design A 2x2x2 factorial arrangement plus an additional positive control treatment (4.6 g kg 21 monocalcium phosphate monohydrate [MCP; Aliphos, Tessenderlo Chemie, Tessenderlo, Belgium] to provide 1.0 g P kg 21 diet) was carried out according to a balanced six rows (periods) by six columns (pigs) design, as given by Shah (1977), which allowed for four replicates per treatment. Experimental variables were Aspergillus niger phytase (Natuphos  , Gistbrocades, Delft, The Netherlands) at doses of 0 and 900 FTU kg 21 , lactic acid in liquid form (85% [wt wt 21 ], purity 98%; Sigma, St. Louis, MO) at doses of 0 and 30 g kg 21 diet and Na phytate (C 6 H 6 O 24 P6 Na 12 , dodecasodium salt from maize, 21 M 5 923.8 g mol , Sigma, St. Louis, MO) in doses of 0 and 7.4 g kg 21 (to provide 1.5 g phytic acid P kg 21 diet). The diets without added MCP, microbial

phytase and Na phytate were formulated to contain 0.7 g digestible P kg 21 of diet at a P digestibility of 24% (CVB, 1996). This amount of digestible P is well below 2.0 g kg 21 of diet (containing 12.5 MJ ME kg 21 ), which is recommended for 40-kg pigs (Jongbloed et al., 1994). There is evidence that an increase in digestible P above recommended levels may lower the percentage of apparent P digestibility ¨ (Schroder et al., 1996). This may mask any stimulatory influences on P digestibility. Thus, the dietary P concentrations were deliberately set below the allowance.

2.2. Experimental diets and feeding Experimental diets were formulated to be isoproteinous (Table 1) and isoenergetic (14.2 MJ ME kg 21 ). The major components were maize and extracted soybean meal, in which intrinsic phytase activity is low ( , 100 FTU kg 21 ), and phytate content relatively high. Manufactering of the experimental diets was described by Kemme et al. (1999). Daily rations were given to pigs twice (at 06:00 and 18:00 h) in a wet, mash form (water:feed ratio 5 2.5 vol wt 21 ). Drinking water and lactic acid were mixed with the feed directly before feeding. Feeding level was 2.3 times maintenance requirement ( 5 418 kJ ME BW 20.75 ), and pigs had no access to drinking water between meals. The levels of ileal digestible essential amino acids were estimated to be 80% of the recommended allowances for a 60-kg growing pig (CVB, 1996). The diets were formulated to contain 6.0 g Ca kg 21 .

2.3. Collection procedures Each experimental period lasted 15 days, including 3 days of gradual diet transition, 4 days of diet adaptation followed by 5 days of faeces collection and, on days 13 and 15, ileal digesta collection. Collected faeces were quantitatively pooled for each animal per period and frozen at 2 208C. Subsequently, faeces were freeze-dried, ground to pass a 1 mm sieve and analysed for ash, total P, Ca, Mg and Cr. Collection of ileal digesta was done according to Kemme et al. (1999). Ileal digesta were freeze-dried, weighed, ground to pass a 1 mm sieve and analysed

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Table 1 Analyzed composition of the experimental diets (g kg 21 )a Diet

1 6b

2 7b

3 8b

4 9b

5

Monocalcium phosphate Microbial phytase Na phytate

1 – –

– – –

– 1 1

– 1 –

– – 1

Maize Soybean meal extract Trace mineral-vitamin premix Cr 2 0 3 -starch premix L-lysine ? HCl L-threonine L-tryptophan Choline chloride (50%) Limestone Maize starch Monocalcium phosphate 1H 2 O Phytase premix (900 FTU g 21 ) Na phytate (C 6 H 6 O 24 P6 Na 12 ) Na carbonate (Na 2 CO 3 )

858.0 112.6 1.4 2.0 2.2 0.2 0.2 0.3 12.7 0.7 4.6 – – 5.1

858.0 112.6 1.4 2.0 2.2 0.2 0.2 0.3 14.7 3.3 – – – 5.1

858.0 112.6 1.4 2.0 2.2 0.2 0.2 0.3 14.7 – – 1.0 7.4 –

858.0 112.6 1.4 2.0 2.2 0.2 0.2 0.3 14.7 2.3 – 1.0 – 5.1

858.0 112.6 1.4 2.0 2.2 0.2 0.2 0.3 14.7 1.0 – – 7.4 –

DM OM CP (Nx6.25) Ash Ca Mg Total P Phytic acid Phytic acid P Phytase activity (FTU kg 21 ) Dietary pH c

863 796 129 37 5.7 1.2 4.0 9.7 2.7 75 6.8

862 828 128 34 5.5 1.1 3.0 9.4 2.6 71 7.3

862 826 130 36 5.9 1.2 4.2 14.0 4.0 957 6.5

862 827 131 35 5.3 1.2 3.0 9.3 2.6 1072 7.3

863 827 129 36 5.7 1.2 4.2 14.3 4.0 61 6.8

a

More details are given elsewhere (Kemme et al., 1999). Diets 6 to 9 were identical to diets 2 to 5, respectively, except that they were acidified with 30 g of lactic acid kg 21 . c The diets 6, 7, 8 and 9 had pH values of 5.4, 5.1, 5.4 and 5.1, respectively. b

for their phytic acid and Cr contents. Samples of the experimental diets were collected and analysed according to Kemme et al. (1999).

2.4. Analytical procedures Dry matter, ash and Kjeldahl N were analysed according to the AOAC (1980). Contents of Ca, Mg and Cr were analysed by atomic absorption spectrophotometry after dry-ashing the samples at 5508C for 4 h. For Ca and Mg analysis, digestion was performed with nitric acid (14.3 M) and for Cr with phosphoric-manganese sulphate solution / potassium bromate solution (Williams et al., 1962). Total P was

determined colorimetrically by the vanadomolybdate procedure (AOAC, 1980). Phytic acid was assayed according to Bos et al. (1991) and phytic acid P content was calculated using the fact that phytic acid contains 28.2% (wt wt 21 ) P. Phytase activity was assayed according to Engelen et al. (1994). The ATTD of ash, total P, Ca and Mg, as well as the AID of phytic acid, were calculated, using Cr as an indigestible marker.

2.5. Statistical analysis Each individual animal was considered as an experimental unit and experimental data were sub-

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jected to analysis of variance with Na phytate, microbial phytase, lactic acid and their interactions, as well as period and animal as factors in the model. The positive control was contrasted with all other treatments. The statistical package Genstat 5, release 3 (Payne et al., 1993) was used. The P-values for the defined contrasts were calculated using Student’s t-test at 0.05 significance level.

slightly lower than expected. Diets 3 and 4, which were supplemented with microbial phytase, had a slightly higher phytase activity than anticipated.

3.2. Apparent total tract digestibility of phosphorus and ileal degradation of phytic acid Supplementation of MCP (1 g P kg 21 ) improved total P ATTD by 7.1 percentage units as compared to the average of all other treatments (P 5 0.023; Table 2). Total P ATTD for the negative control treatment (diet 2) was 20.2%, which was lower (P , 0.001) than in the MCP supplemented diet (diet 1: 43.2%). Total P ATTD in the phytase supplemented diets (diets 3, 7, 4 and 8) was 46.2%, which was not different from that for the diet with MCP addition (data not shown). Total P ATTD was affected by the interaction between lactic acid and microbial phytase (P 5 0.049) and that between Na phytate and microbial phytase (P 5 0.011). There was no significant threeway interaction (P 5 0.465). Addition of lactic acid to the diets without microbial phytase did not enhance total P ATTD. Microbial phytase increased total P ATTD in the absence of lactic acid by 16.2 percentage units, whereas in the presence of lactic acid it improved total P digestibility by 24.3 per-

3. Results Further information on pig performance is described by Kemme et al. (1999).

3.1. Chemical composition of the experimental diets The assayed chemical composition of the experimental diets (Table 1) was close to the calculated composition. In the positive control diet, MCP was supplemented as planned. When Na phytate (7.4 g kg 21 ) was added to Diets 3 and 5, the total P content increased by 1.2 g kg 21 , whereas phytic acid P content was increased by 1.4 g kg 21 . This discrepancy was within the analytical error of phytic acid analysis. Calcium content of the diets was

Table 2 Apparent total tract digestibility (ATTD) of total P and ileal degradation (AID) of phytic acid (%) as influenced by added monocalcium phosphate (MCP) and the interactions of lactic acid 3 microbial phytase and Na phytate 3 microbial phytase (FTU) Interaction lactic acid 3 phytase MCP, g kg Item

0

21

4.6

FTU kg SED B

P-value

21

Lactic acid, g kg 0

30 a

ATTD Total P

AID Phytic acid c A

36.1

43.2

(2–9)

(1)

34.7

2.8

2.85

0.023

0

SED

5.31

, 0.001

0

(1)

900

24.7 (2, 5)

27.3 (6, 9)

40.9 c (3, 4) 10.1 a (2, 5)

51.6 d (7, 8) 20.1 a (6, 9)

50.2 b (3, 4)

P-value

58.4 b (7, 8)

Na phytate, g kg 21 0

7.4 a

0

0

21.5 (2, 6)

30.4 (5, 9)

47.3 c (4, 8) 14.9 a (2, 6)

45.2 c (3, 7) 15.3 a (5, 9)

53.2 b (4, 8)

55.4 b (3, 7)

0.815 900

SED

P-value

2.69

0.011

5.01

0.089

b

0.049 900

5.01 (2–9)

FTU kg 21

b

2.69 900

Interaction Na phytate 3 phytase

21

The treatments that yielded the means are shown between parentheses. SED 5 standard error of difference of means. C The AID of phytic acid was affected by microbial phytase (P , 0.001) and lactic acid (P 5 0.021), but not by Na phytate (P 5 0.729) supplementation of the diet, as main effects. a,b,c,d Different superscripts indicate statistical significance at P , 0.05. B

P. A. Kemme et al. / Livestock Production Science 58 (1999) 119 – 127

centage units. The combined effect of phytase and lactic acid was an increase in total P ATTD by 26.9 percentage units (diets 7 and 8 versus 2 and 5). This stimulatory effect of phytase and lactic acid was equivalent to 0.81 (diet 8 versus 2; 3.0 g total P kg 21 ) and 1.13 g digestible P kg 21 (diet 7 versus 5; 4.2 g total P kg 21 ) for the diets without and with added Na phytate, respectively. Addition of Na phytate to the diets without microbial phytase improved total P ATTD by 8.9 percentage units. Microbial phytase enhanced total P ATTD for the diets without added Na phytate by 25.8 percentage units (diets 3 and 7 versus 2 and 6; 3.0 g total P kg 21 ), this effect being equivalent to 0.77 g digestible P kg 21 . Supplementation of the diet containing microbial phytase with Na phytate did not further enhance total P ATTD. Microbial phytase raised total P ATTD of the diets containing Na phytate by 14.8 percentage units (diets 4 and 8 versus 5 and 9; 4.2 g total P kg 21 ), this rise being equivalent to 0.62 g digestible P kg 21 . There was no significant lactic acid 3 Na phytate interaction with respect to total P ATTD. The AID of phytic acid in the diet supplemented with MCP was lower than in the average of all other treatments (P , 0.001). Phytic acid AID in the negative control treatment (diet 2) was 8.4% (data not shown), which was not different from that for the MCP supplemented diet (P 5 0.454). Phytic acid AID for the diets supplemented with phytase (diets 3, 7, 4 and 8) was as high as 54.3% (data not shown). Lactic acid supplementation of the diets without microbial phytase tended to improve phytic acid AID (P 5 0.08). Microbial phytase stimulated phytic acid

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AID for the diets without lactic acid by 40.1 percentage units, whereas for the diets with lactic acid the rise was 38.3 percentage units. The combined effect of phytase and lactic acid was an increase in phytic acid AID by 48.3 percentage units (diets 7 and 8 versus 2 and 5). This stimulatory effect of phytase and lactic acid was equivalent to degradation of 4.5 (diet 8 versus 2; 9.4 g phytic acid kg 21 ) and 6.9 g phytic acid kg 21 (diet 7 versus 5; 14.2 g phytic acid kg 21 ) for the diets without and with added Na phytate, respectively. Addition of Na phytate to the diets without microbial phytase did not affect phytic acid AID. Microbial phytase enhanced phytic acid AID of the diets without Na phytate by 38.3 percentage units (diets 2 and 6 versus 4 and 8; 9.4 g phytic acid kg 21 ), this effect being equivalent to degradation of 3.6 g phytic acid kg 21 of diet. A similar effect was seen when the diet containing Na phytate was supplemented with microbial phytase. Microbial phytase raised phytic acid AID for the diets with Na phytate by 40.1 percentage units (diets 5 and 9 versus 3 and 7; 14.2 g phytic acid kg 21 ), this rise being equivalent to degradation of 5.7 g phytic acid kg 21 . There was no lactic acid 3 Na phytate interaction as for phytic acid AID (P 5 0.648).

3.3. Apparent total tract digestibility of ash, Ca and Mg No significant treatment interactions were found with regard to ash, Ca and Mg ATTD and, therefore, only the main effects are presented completely (Table 3). Addition of MCP to the diet did not affect

Table 3 Apparent total tract digestibility (ATTD) of ash, Ca and Mg (%) as influenced by added monocalcium phosphate (MCP), lactic acid, microbial phytase and Na phytate a Interactions b

Main effects MCP, g kg 21

Lactic acid, g kg 21

0 (2–9)

4.6 (1)

SED

ATTD Ash Ca Mg

55.8 49.7 25.2

55.9 48.8 26.4

1.55 2.22 2.57

a b c

c

Microbial phytase, FTU kg 21

Na phytate, g kg 21

LA*MP

NaP*MP

P-value

0 (2–5)

30 (6–9)

SED

P-value

0 (2,5,6,9)

900 (3,4,7,8)

SED

P-value

0 (2,4,6,8)

7.4 (3,5,7,9)

SED

P-value

P-value

P-value

0.969 0.693 0.650

53.5 45.3 22.7

58.1 54.1 27.7

1.03 1.48 1.71

, 0.001 , 0.001 0.010

52.2 44.8 22.0

59.5 54.5 28.3

1.03 1.48 1.71

, 0.001 , 0.001 0.002

56.9 49.8 22.3

54.7 49.5 28.1

1.03 1.48 1.71

0.046 0.840 0.004

0.072 0.640 0.749

0.118 0.673 0.465

The treatment(s) that yielded the means are shown between parentheses. LA*MP 5 the interaction between lactic acid and microbial phytase: NaP*MP 5 the interaction between Na phytate and microbial phytase. SED 5 standard error of difference of means.

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ash, Ca and Mg ATTD, as compared to the average of all other treatments. Lactic acid produced stimulatory effects on ash, Ca and Mg ATTD (P # 0.010), the increases being 4.6, 8.8 and 5.0 percentage units, respectively. Microbial phytase had significant effects on the ATTD of ash, Ca and Mg, which were enhanced by 7.3, 9.7 and 6.3 percentage units, respectively. The addition of Na phytate to the diet reduced ash ATTD by 2.2 percentage units, enhanced Mg ATTD by 5.8 percentage units, but left Ca ATTD unchanged.

4. Discussion The main objective of this experiment was to test the hypothesis that the effect of microbial phytase on the ATTD of total P and the AID of phytic acid would become greater when lactic acid is added to the diet. Indeed, this study shows that there was a significant, stimulatory interactive effect of phytase and lactic acid on total P ATTD. Phytase produced an increase in total P ATTD by 16.2 percentage units and lactic acid by 2.6 percentage units. The combination of phytase and lactic acid caused total P ATTD to rise by 26.9 percentage units, i.e., 8.1 percentage units more than the sum of the separate effects of the treatments. Aspergillus niger phytase has its pH optima at 2.5 and 5.5 (Simons et al., 1990) and is therefore mainly active in the stomach (Jongbloed et al., 1992), but a large portion of digesta leaves the pig stomach shortly after feeding and has a suboptimal pH for microbial phytase (Jongbloed et al., 1992). Diet acidification may increase mineral solubility (Jongbloed, 1987), either directly by lowering pH and(or) indirectly by reducing the rate of gastric emptying (Mayer, 1994). Thus, acidification and microbial phytase may have a synergistic effect on P digestibility. Unlike what we expected, there was no interaction between the effects of microbial phytase and lactic acid on phytic acid AID. The lack of interaction may relate to the methodology used. Phytic acid degradation was estimated by the disappearance of inositol hexakisphosphate, so that it is unknown to what extent incomplete degradation had yielded lower inositol phosphates, which still contain P that is

unavailable for absorption. In the absence of microbial phytase, lactic acid produced an increase in phytic acid AID that was much greater than that in total P ATTD. This could imply that the degradation of phytic acid by intrinsic phytase and(or) intestinal phosphatases, as stimulated by lactic acid, is not complete. Thus, after feeding lactic acid, inositol hexakisphosphate may not have been degraded to inositol and inorganic phosphate, but to intermediate inositol phosphates and relatively small amounts of inorganic phosphate. We have estimated the amount of P that was released and apparently digested when the various diets were fed. The amount of phytic acid that has disappeared as caused by the treatments can maximally generate six times as much P on a molar basis. When assuming that all P groups from phytic acid are absorbed and converting the increases in total P ATTD into absolute amounts of digestible P, then the ratio between generated P and phytic acid reflects the fraction of the P groups in phytate that had been hydrolysed. For the diets with lactic acid, but without microbial phytase, the calculated fraction was only 26%, being equivalent to 1.6 P groups in phytic acid. Microbial phytase had a clear stimulatory effect on phytic acid AID, which was equivalent to a release and apparent digestion of 40% or 2.4 of the P groups in phytic acid. Jongbloed et al. (1992) found a greater phytase-induced phytic acid degradation for a maize-soybean meal diet, the percentage of degradation being as high as 79%. However, they used a 1.9-fold higher dose of phytase of a preparation that also contained other enzyme activities. The synergistic effect of microbial phytase and lactic acid was only observed on total P ATTD and not on phytic acid AID, thus it seems that microbial phytase is able to further hydrolyse the inositol phosphates that were partially hydrolysed by lactic acid. The extent of phytic acid degradation was higher in the diets with both additions; 56% or 3.3 of the P-groups were released and apparently absorbed. The addition of MCP to the diet did not affect phytic acid AID when compared with the negative control treatment, which indicates that the level of digestible P supply as such did not influence phytic acid AID. The rise in total P ATTD as caused by MCP addition was similar to that observed in other experiments (e.g., Dellaert et al., 1990). The rise in total P ATTD in the Na phytate

P. A. Kemme et al. / Livestock Production Science 58 (1999) 119 – 127

supplemented diets may be explained by free Na phytate being a substrate with better solubility and accessibility for intrinsic phytase than is the phytate present in feed ingredients. Dietary bound phytate first has to come into solution before it can be hydrolysed. It seems that solubility velocity of phytate in feedstuffs is predominantly governing the action of phytase. The amount of phytic acid in feedstuffs that will come into solution seems to limit the process of phytase action. Phytase will release most of the P-groups in once solubilized phytic acid. This may also explain that the extent of phytic acid degradation in the phytase supplemented diets was always lower in the diets with Na phytate than in the ¨ diets without this supplement. Bruggeman et al. (1962) added different levels of Ca phytate to P deficient diets fed to pigs and observed that on average 25% of phytate P was digested. In our study, 53% of the supplemented phytate P was digested at total tract level in the absence of microbial phytase. The difference between the two experiments with respect to ATTD of P from phytate may also be explained by the higher solubility of Na phytate in comparison with Ca phytate. Microbial phytase generated 0.77 and 0.62 g digestible P kg 21 in the diets without and with Na phytate, respectively. The somewhat lower amount of digestible P generated in the diets with supplemented Na phytate may be related to the high digestible P level (1.9 g kg 21 ) in these diets, implying that the P requirement of the animals weighing 60 kg or more was met or even exceeded. The provision of digestible P above the allowance may have caused a depression of the absorption of P ¨ (Schroder et al., 1996) and was probably not or only partly due to substrate inhibition or relative shortage of phytase. The decreased ATTD of ash that was observed when Na phytate was added to the diet is most likely due to the formation of insoluble complexes between Na phytate and dietary multivalent minerals in the intestinal lumen (Erdman, 1979; Torre et al., 1991). In this experiment, complex formation did not involve Ca and Mg because supplementation of the diet with Na phytate did not affect the ATTD of Ca and even raised that of Mg. Possibly, Na phytate did reduce the bioavailability of Ca, but this effect may have been counteracted by the endocrine control of

125

Ca absorption. The stimulatory effect of Na phytate on Mg ATTD cannot be easily explained. The feeding of Na phytate may have produced a decrease in the pH of the ileal digesta, as also dietary pH was lowered, which would raise Mg absorption (Heijnen et al., 1993). Another surprising effect also emerged from this study as the addition of MCP to the diet was found not to depress Mg ATTD. In general, higher intakes of total P will lower Mg absorption (Windisch et al., 1994), because of the formation of calcium-magnesium-phosphate complexes in the gastrointestinal tract (Brink et al., 1992). However, Pointillart et al. (1983) found a positive relationship between P and Mg absorption. Supplementation of the diet with phytase increased the ATTD of ash, Ca and Mg. Similar effects have been reported earlier (Pallauf et al., 1992a,b; ¨ and Helander, 1994; Lantzsch and Wjst, 1992; Nasi Radcliffe and Kornegay, 1995; Jongbloed and Jongbloed, 1996), the explanation being that microbial phytase degrades phytates and thus releases phytate-bound minerals that become available for intestinal absorption. However, this explanation is not supported by the present observation that the addition of Na phytate to the diet raised Mg ATTD. In general, acidification of the diet has a positive effect on Ca and Mg ATTD as based on studies with formic, butyric, lactic, fumaric and citric acid (reviewed by Jongbloed et al., 1997), but there is no clear relationship between the dose or type of acid and mineral ATTD. The main impact of acidification on digestion of minerals is suggested to be in increasing mineral solubility (Jongbloed, 1987), either directly by lowering pH and(or) indirectly by reducing the rate of gastric emptying (Mayer, 1994). Also, compounds may be formed (at low pH) between the acid group and various cations that act as chelating agents (Ravindran and Kornegay, 1993; Eidelsburger, 1997). Furthermore, acidification may have a positive impact on epithelial cell proliferation in the gastrointestinal mucosa (Sakata et al., 1995), which allows for a more efficient mineral absorption. However, it remains unclear whether the positive effect of organic acids on mineral ATTD derives from a better accessibility of non-complexed minerals or from facilitating the release of phytate-complexed minerals. Lactic acid at a level of 30 g kg 21 of diet raised Ca and Mg ATTD by 8.8 and 5.0

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percentage units, respectively (Table 3), which falls within the range of observations reported earlier for other acids (Jongbloed et al., 1997). We did not observe a synergistic effect of microbial phytase and dietary acidification on Ca and Mg ATTD, which corresponds with earlier work (Radcliffe and Kornegay, 1995; Jongbloed and Jongbloed, ¨ 1996). Lonnerdal et al. (1989) using rat studies and Simpson and Wise (1990) using in vitro studies, found that the affinity of Ca and Zn for inositol phosphates is higher at higher degrees of phosphorylation. Only inositol hexakis- and pentakisphosphate are expected to inhibit mineral absorption because of insoluble complex formation. This would mean that after hydrolysis of two P-groups of phytic acid, mineral absorption is no longer inhibited. Lactic acid probably only partially facilitated the hydrolysis of phytic acid, since on average 1.6 P groups were released. If, as discussed above, microbial phytase further releases the remaining P-groups from partially hydrolysed phytate, no synergism between phytase and lactic acid can be expected. In other words, feeding either lactic acid or microbial phytase will cause that phytate is dephosphorylated to an extent at which the inhibitory effect on Ca and Mg absorption is fully counteracted.

5. Conclusion Supplementation of the maize-soybean meal-based diet of pigs Aspergillus niger phytase at a dose of 900 FTU kg 21 and simultaneous acidification with lactic acid (30 g kg 21 ) resulted in a greater increase in apparent total tract digestibility of total P than was calculated as the sum of the stimulatory effects of the single additions. It is hypothesized that lactic acid delays gastric emptying, which prolongs phytase action in the stomach at its pH optimum. Thus, the combination of phytase addition and diet acidification may be an effective approach to enhance P digestibility in pigs. Evidence is presented that added Na phytate is a better substrate for intrinsic phytase than is intrinsic phytate in feed ingredients. The apparent total tract digestibilities of ash, Ca and Mg and the ileal degradation of phytic acid were also raised by microbial phytase and lactic acid, but no synergism of the treatments could be detected.

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